Project Summary The continued spread of drug-resistant Mycobacterium tuberculosis (Mtb) infections has rendered tuberculosis (TB) a global health pandemic and pressed home the urgent need for new drugs. Compounds which inhibit essential components of the Mtb cell wall rapidly cause cell death and are represented clinically by isoniazid. If any drug combination is likely to reduce treatment duration and prevent the emergence of new antibacterial resistance, it will be a sterilizing synergistically acting therapy. In this proposal, our aim is to develop a new TB therapy that targets a validated druggable pathway and commences with a validated lead compound. The long- term goal is a new drug effective against both drug-susceptible and drug-resistant Mtb. The drug lead DG167 targets KasA, an essential enzyme involved in mycolic acid biosynthesis. The atomic-scale interactions between KasA and DG167 and multiple analogs of differing chemotype have been established in high-resolution X-ray crystal structures. Our work corrects a previous GSK structure and shows that two molecules of DG167 are bound in KasA. DG167 shows synergy in killing studies with the established drug isoniazid. DG167/isoniazid combinations lead to rapid sterilization of Mtb cultures and show synergy in a mouse model of acute Mtb infection. A DG167/isoniazid combination could shorten TB treatment by accelerating clearance of replicating bacteria in acute TB disease. Here, we propose to advance lead optimization of DG167. Our experienced project team is comprised of academic and industrial researchers with an established track record of collaboration and the common goal of devising new antitubercular drug therapies. The complimentary key skill sets in medicinal chemistry, cheminformatics, structural biology, microbiology, and pharmacokinetics/pharmacodynamics are all present and will be leveraged to deliver an optimized lead candidate inhibitor of Mtb KasA with enhanced in vivo efficacy as compared to the early lead DG167. This will prepare us for a Phase II program that would complete the optimization and set the stage for pre-IND toxicology studies.